Ion sensor based on differential measurement, and production method

ABSTRACT

Ion sensor based on differential measurement comprising an ISFTET-REFET pair wherein the REFET is defined by a structure composed of an ISFET covered by a microreservoir where an internal reference solution is contained. The sensor comprises a first and a second ion-selective field effect transistor, an electrode, a substrate on the surface whereof are integrated the two transistors, connection tracks and the electrode and a structure adhered on the first ion-selective field effect transistor which creates a microreservoir on the gate of said first transistor, with the microreservoir having a microchannel which connects the microreservoir with the exterior and the microreservoir being filled with the reference solution.

OBJECT OF THE INVENTION

The present invention relates to an ion sensor based on differentialmeasurement and to the manufacturing method thereof. Said sensorperforms the measurement of the concentration of certain ions in asolution using ISFET (ion-selective field effect transistor) transistorsand it compares said measurement with that of a reference solution whichis stored in a microreservoir, performing said measurement via an ISFETtransistor whose gate is kept in contact with said reference solution,also called REFET transistor (field effect transistor which does notrespond to the ion concentration), and which, therefore, has zeroresponse to the ions which are to be measured.

The technical field wherein the present invention is included is that ofthe sector of physical technologies and its more typical application isfor ion measurement, for example, pH (concentration of hydrogen ions ina solution), in various sectors such as the food industry andbiomedicine.

BACKGROUND OF THE INVENTION

In the current state of the art, the measurement of concentrations ofvarious ions of a medium is performed in highly varied ways. One of themost widely used techniques is the use of test strips. These test stripsare strips of paper with different areas which become coloured incontact with aqueous solutions, taking on different colours depending onthe concentration of specific ions of the measurement solution. Toidentify the concentration of ions of the solution, after wetting thestrip therewith, the user must compare the colours obtained with thoseof a table provided by the manufacturer. The result of this measurementtechnique greatly depends on the correct manipulation by the user andfactors such as: the presence of proteins in the samples, the reactiontime of the strip with the samples, or the homogeneity of the samples.An incorrect manipulation generates many false results (positive andnegative). Furthermore, it is generally considered that the resolutionof this technique is of 0.5 units, for the specific case of pHmeasurement, which lacks sufficient diagnostic value to take clinicaldecisions in some biomedical applications such as in Urolithiasis (KwongT. et al. “Accuracy of urine pH testing in a regional metabolic renalclinic: is the dipstick enough?Urolithiasis 2013).

The standard measurement technique of ion concentration is atomicabsorption. However, this technique requires a complex installation andits miniaturization is not feasible.

Ion Selective Electrodes (ISEs) are used for simpler measurements interms of equipment and are less expensive. These electrodes have aselective membrane so that, by the exchange or interaction of thesolution ions with the membrane, the ion activity becomes an electricpotential. The selective membrane may be of several types, of glass,crystalline or based on ion-exchange compounds. The latter have apolymer (e.g. polyvinyl chloride, PVC) which immobilizes the ionselective compound. The measurement of the electric potential of theISEs requires the use of a reference electrode, which is frequentlyintegrated in the very body of the ISE (combined electrodes). Thereference electrode is generally a metal electrode immersed in areference solution which is in turn connected to the solution to bemeasured through a liquid bond. The main characteristic of the referenceelectrode is that its potential, i.e. the potential between the interiorof the metal and the inside of the solution wherein it is immersed, doesnot depend on the composition of said solution. The reference electrodesusually have losses of reference solutions through the liquid bond, sothat a periodic refilling thereof is required.

To obtain precise measurements, these electrodes require a priorcalibration which consists of the measurement of the potential generatedwhen the electrode is immersed in a known ion concentration solution.These electrodes form part of an instrument, which in the case of the pHis known as pHmetre, which is not cheap to manufacture, nor is itportable or autonomous and it requires specific maintenance and cleaningconditions for its correct conservation. The result of this measurementtechnique also depends on the correct manipulation by the user (who mustbe suitably trained for said purpose). An incorrect manipulation orconservation of the electrodes may give rise to false results.

Another type of sensors used for this class of measurements are theISFET (ion-selective field effect transistor)-type sensors. These aredevices manufactured using microelectronic technology. The potential ofthe solution (which is the transistor gate potential) is controlled by areference electrode such as those used for the measurement with ISE-typeelectrodes. The ISFET is a field effect transistor whose thresholdvoltage varies with the ion concentration of the solution in contactwith its gate dielectric. For many types of dielectrics (SiO2, Si3N4,Al2O3, Ta2O5, ZrO2), the variation of threshold voltage of the ISFETmainly depends on the H⁺ ion and, therefore, it is used as pH sensor. Tomake sensors of other ions based on the ISFET, an additional layercalled selective membrane as disclosed in U.S. Pat. No. 5,250,168 isdeposited on the gate dielectric layer. Depending on the membranedeposited, the ISFET would function as a sensor for specific ions orothers. The measurement with these sensors consists of recording thechanges in threshold voltage of the field effect transistor, which areproportional to the changes in ion concentration that one wants tomeasure. A way of measuring the changes in threshold voltage of theISFET is using a circuit which polarizes the device with a constantdrain current and a constant source drain voltage. In this way, thechanges in gate voltage the circuit applies to maintain saidpolarization are equal to the threshold voltage changes suffered by theISFET. Therefore, the gate voltage applied by the circuit is taken asoutput signal.

Both the measurement systems based on ISE electrodes and those based onISFET require a reference electrode to be able to measure the ions. Thismakes them expensive and requires periodical maintenance. In 1978, asolution was disclosed for pH measurement with ISFET-type deviceswithout reference electrode (P. A. Comte and J. Janata, “A field effecttransistor as a solid-state reference electrode”, Analytica ChimicaActa) which consisted of the differential measurement of an ISFET and aREFET. In this case the REFET is formed by an ISFET the gate whereof iskept exposed to a constant pH. The differential measurement consists ofmeasuring the threshold voltage of both devices using a single electrodeimmersed in the solution as terminal gate and obtaining the answer assubtraction of the two values obtained. The REFET gate is maintainedexposed to a constant pH by the incorporation of a microreservoir filledwith reference solution (internal solution). Said microreservoir isconnected to the exterior via a microchannel which acts as liquid bond,so that the difference in potential between the external solution andthe reference solution is small and is not greatly influence by the pHor the concentration of other ions in the external solution. In thisway, the changes in potential which occur between the electrode and thesolution are transferred to both threshold voltage values, and thereforehave no impact on the differential value (they are cancelled in thesubtraction operation). For this reason, the different measurementsystem can be implemented with any conductor electrode, without the needfor it to be reference. Given that the REFET is exposed to a constant pHsolution, the variation in the differential value shall be equivalent tothe ISFET's response to the change in pH. However, the way ofmanufacturing the REFET described by Compte and Janata is difficult toautomate and would therefore not make it possible to manufacture thesensors at a cost greatly less than those of the ISFETs with referenceelectrode, which would not allow its price to be accessible to thegeneral public. Furthermore, in the design of the ISFET-REFET sensordescribed by Compte and Janata, the REFET's microreservoir isconstructed with an epoxy resin. This microreservoir, once the resin hasbeen cured, is filled with an agarose gel prepared in a buffer solution.Subsequently, a glass capillary is introduced, which acts as amicrochannel, in the agarose gel and the microreservoir is sealed with alayer of epoxy resin. Thus, the sensor is stored dry; the bufferedsolution of the microreservoir is slowly evaporated through themicrochannel, being replaced by air. The presence of air interior themicroreservoir prevents it functioning correctly when it is used afteran extended time has passed of immersion in aqueous solution. This isdue to the fact that the filling with water, as well as the necessarydiffusion of the trapped air to the exterior, is solely performedthrough the microchannel, which is not filled with hydrogel.Furthermore, the lifetime of this type of sensor depends on the volumeof the microreservoir and on the dimensions of the microchannel whichconnects it with the exterior, since the reference solution in themicroreservoir shall be diluted and contaminated through themicrochannel, so that the error in the measurement may progressivelyincrease a measurement that the pH of said solution varies with respectto its original value. For this reason, it is considered a sensor with ashort lifetime.

Document EP 85200263 discloses a sensor wherein use is made of two ISFETsensors, one of which is found interior a conduit wherethrough thereference solution flows. In this way, said ISFET is always in contactwith an uncontaminated solution. However, for this it is necessary toincorporate in the sensor a reference solution injection system as wellas the means of supply of the injection system which make the solutiondescribed more complex and expensive.

Thus the state of the art has the following associated problems: thetest strips are imprecise; the glass electrodes are expensive, fragile,require maintenance and cannot easily be miniaturized; the current ISFETand ISE-type sensors are miniaturizable but are expensive and requiremaintenance as they must be used with a reference electrode; theISFET-REFET sensor proposed by Compte and Janata is expensive tomanufacture and has a short lifetime; and the sensor disclosed in EP85200263, in addition to concerning two ISFET transistors, has a greatercost and complexity due to the need to have a reference solutioninjection system.

DESCRIPTION OF THE INVENTION

The present invention discloses a novel ion sensor based on differentialmeasurement comprising at least one ISFET transistor and a REFETtransistor. The REFET is defined by a structure composed of an ISFETcovered by a microreservoir where an internal reference solution iscontained. A second object of the present invention is the manufacturingmethod of the sensor previously described which allows its massproduction at low cost. A third object of the present invention is thesensor previously described immersed in a conditioning receptacle filledwith the reference solution, which makes it possible to extend theuseful life of said sensor.

Thus the first object of the present invention is an ion sensor based ondifferential measurement. Said sensor is characterized in that it atleast comprises:

a first ion-selective field effect transistor and at least one secondion-selective field effect transistor, electrically connected byconnection tracks to a measurement circuit;

an electrode of a conductor material;

at least one chip on the surface whereof are integrated the two fieldeffect transistors. The chips shall preferably be of a semiconductormaterial;

a structure coupled on the first transistor configured to create amicroreservoir on the gate of said first transistor and at least onemicrochannel which connects the microreservoir with the exterior, themicroreservoir and the microchannel being filled with a referencesolution. This structure adhering to the first field effect transistoris what creates the REFET; and,

a substrate whereon are integrated the chips, the connection tracks andthe electrode;

an encapsulating material which electrically insulates the connectiontracks and partially insulates the first and second transistor of thesolution to measure. This encapsulating material avoids that a flow ofelectrical current is produced between the solution (which together withelectrode form the gate terminal) and another terminal of the transistor(drain, source or substrate).

It has been provided, in a particular embodiment of the invention, thatthe sensor described here integrates a single REFET and a plurality ofselective ISFETs, each one of them to a different ion. This is achievedby having a series of selective membranes disposed in each ISFET so thateach one of them detects a different ion. Both the ISFET and the REFETmay be in the same chip or different chips, but all the ISFETs performthe differential measurement with respect to the same REFET. Thus it ispossible toachieve, with a single sensor, a plurality of measurements ofconcentrations of different ions at the same time.

In a particular embodiment of the invention, the reference solution iscontained in a hydrogel which occupies the volume of the microreservoirand the microchannel.

The REFET is preferably constructed from a H+ ion selective ISFET, withthe reference solution being a buffer which fixes the pH at a determinedvalue, but it also proposes that the REFET is constructed from an ISFETselective to another ion, i.e. that it includes a membrane selective tosaid ion on its gate dielectric, in which case the reference solutionmust contain a determined concentration of said ion.

In another particular embodiment of the invention, the firstion-selective field effect transistor is integrated in a first chip andat least one second ion-selective field effect transistor is integratedin a second chip. If there is a plurality of second transistors, eachone of them could be integrated in an independent chip or they could allbe integrated together in a single chip.

In another particular embodiment of the invention, it has also beenprovided that the first ion-selective field effect transistor and the atleast one second ion-selective field effect transistor are integrated inthe same chip. Thus it manages to reduce the manufacturing time andcosts of the sensor. In another particular embodiment of the invention,the field effect transistors, the connection tracks, the electrode and apart of the measurement circuit are integrated in the same chip. Thus itmanages to further reduce the manufacturing costs of the sensor andreduce its size considerably, which may be important for certainapplications.

In another particular embodiment of the invention, the connection of thechips, more specifically of the connection “pads” of the chips, with theconnection tracks is performed via wire bonding.

In another particular embodiment of the invention, the chips areencapsulated by a polymer, with the wires and the connection tracksbeing covered by said polymer and the gates of the first and secondion-selective field effect transistor, the outlet of the microchanneland the electrode being uncovered.

In another particular embodiment, it has been provided that the outerwalls of the microreservoir of the first transistor (REFET) are, atleast partially, of a material permeable to water molecules in gas phaseand to air but not to the solution with the reference pH. Thus, thediffusion of the air molecules towards the exterior and of the watermolecules towards the interior is performed throughout the surface ofsaid permeable material accelerating the tuning process of the sensorwhen it is going to make use of it after a period in which it has notbeen used. In this way, it allows the dry storage of the sensor and afast rehydration of the microreservoir before its use by the immersionin a conditioning solution. This obviously very considerably lengthensthe useful life of this type of sensors.

In another particular embodiment of the invention an ion-selectivemembrane is placed on the gate of the at least one second transistor(ISFET). Thus, depending on what type of ions the membrane placed isselective to the ion sensor based on differential measurement object ofthe present invention can measure concentrations of different ions. Inthis way it is possible to obtain sensors to measure concentrations ofdifferent ions such as Ca2+, K+, Na+, Cl, NH4+ or CO32−.

In another particular embodiment of the sensor object of the presentinvention, the microreservoir has a volume between 0.001 mm³ and 1 mm³and the microchannel has a section of between 1 square micrometre and10000 square micrometres and a length between 10 microns and 1 mm. Theconcentration of chemical species interior the microreservoir follows anexponential evolution as these species diffuse through the microchanneltowards the exterior. The time constant of that concentration variationis proportional to the section of the microchannel and inverselyproportional to the volume of the microreservoir and the length of themicrochannel. Therefore, the time taken to lose a certain quantity ofthe chemical compounds of the buffer which maintain the concentration ofions of the solution in the interior of the microreservoir and thedegree of contamination of said solution with compounds from theexterior, is proportional to the section of the microchannel andinversely proportional to the volume of the microreservoir and to thelength of the microchannel. In other words, a longer and/or finermicrochannel provides a stable sensor signal during a longer time.However, a longer and finer channel also involves a greater electricalresistance of the microchannel filled with solution. As the microchannelmust electrically connect the solution of the interior of the reservoirwith the solution of the exterior to transmit the potential of theelectrode to the REFET transistor gate, the greater the resistance ofthe microchannel, the greater the susceptibility of the sensor toelectrical interference. This limits the dimensions of the microchanneland therefore the stable measurement time that can be obtained with thegiven dimensions of the microreservoir.

In another particular embodiment of the invention, it has been providedthat there are 2 or more microchannels that connect the microreservoirwith the exterior. Increasing the number of microchannels may allowreducing the section thereof without increasing the electricalresistance between the solution of the microreservoir and the solutionto measure. A sufficiently reduced section of the microchannels avoidsthe intake of certain microorganisms in the interior of themicroreservoir which could alter the characteristics of the referencesolution of the surface of the REFET gate dielectric.

In another particular embodiment of the invention, it has been providedthat removable and external sealing means are disposed, such as, forexample, adhesive tape or similar, in the outlet of the microchannel toseal the content of the reservoir and the microchannel. The adhesivetape has the suitable form to be able to be manually removed. This makesit possible to lengthen the useful life of the sensor since the solutionwithin the REFET is insulated, avoiding it from evaporating, until thefirst use of the sensor. Additionally, the material from which thestructure which creates the microreservoir has been manufactured shallnot be permeable to the reference solution.

In another particular embodiment of the invention, the structure whichcreates the microreservoir is at least partially of a gas permeablepolymer, such as, for example, polydimethylsiloxane, which makes itpossible to store the sensor dry allows using it after a few hours ofsoaking. This advantage is important to facilitate the storage andmarketing of the sensor or to facilitate the transport thereof in theevent that it is used in a portable measuring apparatus.

A second object of the present invention is the manufacturing method ofthe ion measurement sensor based on differential measurement describedabove. Said method at least comprises the following phases:

-   -   disposing equally spaced a plurality of first ion-selective        field effect transistors on a first wafer;    -   coupling, preferably by bonding, a structure of bondable        material on the first wafer, creating a plurality of equally        spaced microreservoirs and microchannels in correspondence with        the first ion-selective field effect transistors, so that each        microreservoir is situated in correspondence and aligned with        each first ion-selective field effect transistor;    -   cutting the first wafer in chips, where each chip comprises a        first field effect transistor and a structure with a        microreservoir and at least one microchannel;    -   bonding on a substrate the first ion-selective field effect        transistor with the microreservoir and the microchannel, a        second ion-selective field effect transistor, the electrode and        the connection tracks;    -   connecting the connection tracks to the first and second        transistor and encapsulating said first and second transistor        and the connection tracks.

For the case wherein the first and second ion-selective field effecttransistor are integrated in the same chip, the second wafer of bondablematerial has at least one orifice in proximity to each microreservoir,so that each orifice during the bonding phase is placed incorrespondence with the gate of a second field effect transistor leavingexposed to the exterior said gate of the second transistor so that it isin contact with the solution to measure.

In a particular embodiment of the method object of the presentinvention, the coupling phase of the structure on the first wafercomprises adding a plurality of layers of bondable material previouslysubjected to a photolithography process on the first wafer, to generatethe structure with the microreservoir and the microchannels.

In another particular embodiment of the method object of the presentinvention, the coupling phase of the structure on the first wafercomprises previously subjecting the structure of bondable material to arecessing process for the creation of the microreservoirs and themicrochannels. This recessing process may be by vacuum, extrusion orsimilar of a second wafer.

In another particular embodiment it has been provided that themicrochannel is integrated in the chip by a longitudinal recessing inthe surface of the first wafer, i.e. of the chip. In this way, thestructure of the REFET is completed by the bonding of a second wafersince it only contains now the reservoirs, or by the addition of layersof bondable and photolithographiable material on the ISFETs, to form thewalls of the microreservoir and the covers of said microreservoirs.

It has been provided to deposit a layer of insulating material on thesurface of the first and second field effect transistor to insulate thedrain and the source of the first and second field effect transistor andthe substrate of the solutions. Thus, only the gate of the first andsecond field effect transistor remains in contact with the solutions,both reference and that in which one wants to know the ionconcentration. In another embodiment of the invention, encapsulatingmaterial is additionally deposited on all the edges of the first andsecond ion-selective field effect transistor to electrically insulatethe substrate of the first and second ion-selective field effecttransistor of the solution to measure.

Alternatively, it is possible to use a wafer structure which providesthe insulation so that said transistors are also electrically insulatedfrom the substrate without the need to use encapsulating material in itsedges. For example, it is possible to use SOI wafers (thin layer of asemiconductor on an insulating layer) to form the two field effecttransistors thereon. To obtain the insulation, once the transistors havebeen formed, a trench must be made in the semiconductor layer whichtotally surrounds each one of the transistors, after depositing theinsulating layer and finally eliminating the insulating layer from thegate of the transistors and of the wire bonding areas (connection pads).Another form of obtaining insulation is forming the transistors within aregion of semiconductor insulated from the rest of the substrate by ap-n junction. In this case, it is necessary to guarantee that the p-njunction is inverse, i.e. that the potential of the p region is morenegative than that of the n region.

The manufacturing method of the ion sensor based on differentialmeasurement described here has the advantages, compared with the stateof the art, that it can be more easily automated and executed on a largescale, and therefore, allows a considerable reduction in themanufacturing costs thereof.

A third object of the present invention is a conditioning receptacle tostore the aforementioned sensor between measurements which makes itpossible to extend the lifetime of the sensor indefinitely. Theconditioning receptacle will be filled with the reference solution,which allows the solution contained in said microreservoir to be renewedby diffusion through the microchannel.

Since the chips that contain the transistors are encapsulated in asurface which contains metal connection tracks, it is easy to add othercomponents to the sensor (by bonding on the tracks). Some examples wouldbe: 1) A transient voltage suppressor to protect the transistors fromelectrostatic discharges, e.g. connected between the electrode and thetransistor substrate terminal, 2) a thermistor to measure temperatureand compensate the thermal drift of the sensor, 3) a memory to storesensor parameters, for example, of the sensitivity to ions and thecoefficients of variation with the temperature of each sensor, 4) apolarization and measurement circuit of the ISFET-REFET pair, 5) ananalogue-digital converter, 6) a microcontroller, 7) a display to showthe measurement data, 8) an interface circuit to communicate the data bya serial protocol (for example, USB standard) with an electronicapparatus (for example a computer or smartphone), 8) a battery, 9) acommunications circuit and an antenna to communicate the data wirelesslyto another electronic apparatus. A particular combination of thesecomponents would give rise to an ion sensor of RFID (RadiofrequencyIdentification)-type. In this case, the ISFET and the REFET areintegrated with a measurement circuit and an analogue-digital converterand with the rest of the circuitry and components typical of a RFID tag.This would make it possible to use a RFID tag reader to obtain the ionmeasurement data from the exterior of a closed receptacle, the RFIDsensor being immersed in the liquid to measure interior the receptacle.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1.—Shows a sectional view of a particular embodiment of the ionsensor based on differential measurement object of the presentinvention.

FIG. 2.—Shows a sectional view of the particular embodiment of the ionsensor shown in FIG. 1 whereto sealing means of the microchannel and themicroreservoir have been added.

FIG. 3.—Shows a particular embodiment of the manufacturing method of theion sensor based on differential measurement, object of the presentinvention. FIG. 3a shows the alignment phase of both wafers. FIG. 3bshows the bonding phase of both wafers. FIG. 3c shows the filling phaseof the reservoirs with solution or hydrogel. FIG. 3d shows the cuttingphase of the resulting wafer into chips.

FIG. 4.—Shows a particular embodiment of an alternative manufacturingmethod of the ion sensor based on differential measurement, object ofthe present invention. FIG. 4a shows the ISFET. FIGS. 4b to 4g show thesubsequent bonding phases of layers of polymer alternated withphotolithography phases to configure the microreservoir and themicrochannel.

FIG. 5.—FIG. 5a shows a plan view of an example of embodiment of thesensor with all its components. FIG. 5b shows the sensor of FIG. 5awherein the encapsulating polymer has been added.

FIGS. 6a -6b.—Shows an example of the use of the ion sensor of FIG. 1 inthe measurement of a determined ion concentration of any solution.

DESCRIPTION OF AN EXAMPLE OF EMBODIMENT OF THE INVENTION

Below, an example of embodiment of the invention is described withillustrative and non-limiting character, making reference to thenumbering adopted in the figures.

FIG. 1 shows an example of embodiment of the ion sensor based ondifferential measurement, object of the present invention, for thespecific case wherein it has been designed for measurement of the H⁺ion, i.e. for the case wherein the pH of a specific solution is to bemeasured. Said sensor is formed by an ISFET (1) and a REFET (2), wherethe REFET (2) is composed in turn of another ISFET (3) the gate (4)whereof is maintained exposed to a constant pH by incorporation of astructure (5) which creates a microreservoir (6) filled with a referencesolution (internal solution) with a constant pH. Said microreservoir (6)is connected to the exterior by a microchannel (7). This microchannel inthis specific example of embodiment comprises being formed by twosections thereof perpendicular to one another, but it could be formed bya single longitudinal section or have any other configuration.

Both the ISFET (1) and the REFET (2), both integrated in differentchips, are in turn fixed on a substrate (8) which has a metal layerdefined in the form of connection tracks (9) and an electrode area (10).The chips are partially encapsulated using “chip-on-board”-typetechniques, i.e. connection by wire-bonding (soldering of the connectionpads (14) of the chips by wire (12)) and protection with glob-top(encapsulating polymer (11)). The encapsulating polymer (11) covers theconnection wires (12) and the connection tracks (9) and leaves uncoveredthe gates of the ISFET (13) and the REFET (4) of, at least partially,the structure (5) which creates the microreservoir (6) and, completely,the outlet of the microchannel (7) of the REFET (2), as well as theelectrode area (10).

The REFET chip (2) is formed by the ISFET chip (3) with the structure(5) adhered to its surface forming the microreservoir (6) on the gate(4) of said ISFET (3) and the microchannel (7), so that the walls andthe ceiling both of the microreservoir (6) and of the microchannel (7)are of the material of said structure (5), whilst the floor is formed bythe surface of the ISFET (3).

FIG. 2 shows a specific example of embodiment wherein sealing means ofthe microchannel (7) and of the microreservoir (6) have been adhered tothe microchannel (7) outlet. Specifically, an adhesive strip (15) hasbeen adhered, which has a portion without adhesive material so that itcan be easily removed by a user. This particular embodiment makes itpossible to lengthen the useful life of the sensor since the referencesolution in the microreservoir (6) and in the microchannel (7) of theREFET (2) is completely insulated avoiding leaks or the evaporationthereof until the time wherein the sensor is going to be used for thefirst time and the adhesive strip (15) is removed.

FIG. 3 shows an example of embodiment of the manufacturing method of thesensor described herein. The method is based on the formation of themicroreservoir and the microchannel and its bonding on the ISFET usingplanar technology processes, such as those used for the manufacturing ofmicrofluidic systems. FIG. 3A shows a first wafer (16) where previouslyand equally spaced it has integrated ISFETs, (20) and connection pads(14) and a second wafer (17) with microreservoirs (18) and microchannels(19) previously made in its lower surface. This second wafer (17) is ofa material which means it can be bonded to the first wafer (16). Bothwafers (16,17) are aligned so that a microchannel (19) and amicroreservoir (18) corresponds to each ISFET (20). The phase whereinthe bonding between both wafers (16,17) is performed is shown in FIG.3B.

In this sense and for the bonding phase between both wafers (14,15), thefollowing process has been provided. The bonding is preferably ofchemical type, i.e. by means of the functionalization of the surfaceswith molecules which react forming covalent bonds, but other bondingtechniques, provided that the geometry of the microchannels (19) is notdistorted. Many combinations of materials are known in the state of theart which may be functionalized and chemically bonded. In this regard,one possibility is that the first wafer (16) has the surface of siliconoxide or oxynitride and the second wafer (17) is of polydimethylsiloxane(PDMS), both functionalized by means of an oxygen plasma.

The second wafer (17) is of easy manufacturing with micro-manufacturingtechniques used for the implementation of microfluidic systems. It isprovided that it has the structure already formed by moulding or someother technique.

The ISFETs (20) are manufactured with a technology that allows them tobe insulated from the substrate (8). Said technique is based on the useof SOI wafers and the definition of insulation trenches around theISFET. In this way it facilitates the encapsulation since it is nolonger necessary to protect the chip edges. This allows encapsulatingchips with a more reduced area, since the distance of the ISFET gate tothe chip edges is no longer critical as there is no danger of the gatebeing accidentally covered when the encapsulating polymer is applied.

After cutting the wafers (16, 17) into individual chips (21), as shownin FIG. 3D, they can be encapsulated by automatic techniques similar tothose established in the microelectronic industry such as “chip onboard”. This consists of adhering the chips (21) to a substrate(typically a printed circuit board), connecting them by wire bonding(12) and protecting the assembly with an encapsulating polymer (11). Thevariation in this case with respect to the standard technique is thatthe encapsulating polymer (glob-top) does not cover the entire chip, butit is only applied on the area of the connection wires. The sensor withall its components already assembled on the substrate is shown in FIG.3D.

FIG. 4 shows another alternative manufacturing technique of the REFETwherein a REFET is obtained by layer addition starting from a chip wherean ISFET is integrated. FIG. 4a shows an ISFET on a chip (22) whereinsaid ISFET comprises a source pad (23) connected to the source (27) ofthe transistor, a drain pad (25) connected to the drain (28) of thetransistor and a substrate pad (24) (all of them form the connectionpads (14) of the ISFET) and a gate (26). Subsequently, a first layer(29) of polymer is deposited by bonding or a pre-polymer is deposited bycentrifugation and is then heat cured, as shown in FIG. 4b . This layerof polymer (29) is structured (FIG. 4c ) by lithography creating themicrochannel (30) and the microreservoir (31) and leaving the connectionpads (23,24,25) free of polymer. Subsequently, and as shown in FIG. 4d ,a second layer (32) of polymer is bonded by lamination on the firstlayer (29) of polymer. Again, this second layer (32) of polymer isstructured by photolithography increasing the volume of themicroreservoir (31) and closing the microchannel (30) (FIG. 4e ) butleaving free the outlet orifice (33) of the microchannel (30). Finally,a third layer (34) of polymer is bonded and it is structured bylithography (FIGS. 4f and 4g ) thus the microreservoir (31) remainsclosed and only the outlet orifice (33) of the microchannel (30) remainsopen. The three layers of polymer (29,32,34), which may be SU8, definethe microreservoir (31) and the microchannel (30) which in turn isconnected to the exterior through its outlet orifice (33). This outletorifice (33) allows the filling to wafer level of the microchannel (30)and the microreservoir (31) with hydrogel or with any referencesolution.

The structure of an ISFET is similar to that of a MOS transistor(diffusion of drain and source in a doped semiconductor substrate) withthe difference that it does not have a gate electrode and the gatedielectric is exposed. So that the ISFET and REFET devices functioncorrectly they must have the gate dielectric in contact with thesolution, the ISFET gate dielectric with the solution that one wants tomeasure and the REFET gate dielectric with the reference solution, butthey must have the drain, the source and the substrate insulated fromthe respective solutions. To guarantee this, a layer of insulatingmaterial is deposited on the chip surface during its manufacturing (atwafer level), and the chip edges are protected with the encapsulatingpolymer during the encapsulating process. Alternatively, it is possibleto use a manufacturing technology that makes it possible to electricallyinsulate the device substrate from the chip edges, so that it is notnecessary to protect them with polymer, for example using SOI (Siliconon insulator) wafers. To be able to use the “chip-on-board” standardencapsulation technique it is possible to use the two ways of insulatingthe substrate, but the first requires a large space (˜2 mm) between theISFET gate and the edge of the chip in all directions, which makes itnecessary to have large, and therefore expensive, chips. The secondoption, via the se of SOI wafers becomes more suitable for themanufacturing of the ISFET-REFET sensor described herein, since itallows the encapsulation of chips of reduced area, only requiring thatthe separation is large in one direction (for example, in a rectangularchip the ISFET gate would be situated at one end of the chip and theconnection pads to protect with glob-top at the other end of the chip).

An interesting variant of the REFET is the one where microreservoir andthe microchannel are filled with a hydrogel. The advantages in this caseare of avoiding problems of bubble formation in the microchannel and themicroreservoir (which could cause malfunctioning) and the possibility ofstoring the sensor dry until its use. The hydrogel would be soaked inthe reference solution and would perform the same function as theinternal solution without hydrogel. This material is very hygroscopic,so that it would take a lot longer to dry if the sensor was left outsidethe solution. In the event of totally drying, it could be easilyrehydrated by re-immersing in distilled water or in reference solutionwithout the danger of bubbles forming.

FIG. 5 shows a plan view of an example of embodiment of a pH sensor inaccordance with the present invention. FIG. 5a shows a PCB substrate(35) wherein an ISFET and a REFET such as those described in FIG. 4, anelectrode (36) and connection tracks (37) have been fixed. Said tracksare connected both to the ISFET and the REFET by the connection pads(23,24,25) by wire bonding (38). FIG. 5b shows the sensor of FIG. 5awherein the encapsulating material (39) has been deposited, whichpartially covers both the ISFET and the REFET and totally covers theconnections thereof with the connection tracks (37).

Finally, it has been provided to integrate in a single chip the ISFETand the REFET and even further reducing the sensor's cost.

Another object of the invention is also an ion measurement method usingan ISFET/REFET sensor described. When it is not being used, the sensor(40) is introduced in a conditioning receptacle (41) filled with areference solution (42) (FIG. 6a ). This reference solution (42) alsoserves as calibration solution as its ion concentration is known. Oncethe sensor (40) has been introduced for the first time in the referencesolution (42) and sufficient time has been given so that themicroreservoir (6) fills or is soaked with said solution (42), saidsensor (40) is removed from the conditioning receptacle (41), it isrinsed and immersed in the solution to measure (43) situated within ameasurement receptacle (44), keeping the microreservoir (6) of the REFETfilled with the reference solution (42) (FIG. 6b ). After its use, thesensor (40) is cleaned and is re-inserted in the conditioning receptacle(41) so that the microreservoir (6) solution is balanced with areceptacle solution and returns to its original ion concentration. Thesensor (40) would function correctly whenever the time of use is lessthan the time wherein the sensor (40) is immersed in the referencesolution (42) within the conditioning receptacle. The present inventionis novel in that the sensor (40) is maintained in the conditioningreceptacle (41) between one measurement and the next, which means thatthe sensor does not have a limited lifetime due to contamination of thereference solution or of diffusion of its components towards theexterior. An added advantage is that as the conditioning receptacle (41)is filled with the reference solution (42), whose ion concentration isfixed (for example a buffered solution to maintain the constant pH inthe event that the ISFET is selective to the pH and the REFET isconstructed with a pH-selective ISFET), the sensor can be calibratedbefore its removal in the same way transparent to the user.

Among the multiple applications that can be given to the ion sensorobject of the present invention is that of integrating the sensor in aself-diagnostic medical device by the measurement of, for example, ionsin urine, which may be of interest for controlling diseases such aslithiasis and osteoporosis. Another possible application would be themeasurement of vaginal pH for birth control, where the measurement madeby the sensor was transmitted to a mobile device (for this the sensorobject of the present invention must have a communication interface withthe mobile device). Another possible application for the sensor would bethe monitoring of ions in cell cultures. Introducing the sensor withinthe culture medium, it would be possible to continuously control thestate of the cells without the need to open the receptacle lid. In thiscase the measurement could be transmitted to a wireless communicationsystem integrated in the sensor.

1. Ion sensor based on differential measurement, characterized in that it comprises: a first field effect transistor and at least one second ion-selective field effect transistor, electrically connected by connection tracks to an ion measurement system, the second ion-selective field effect transistor being in contact with a reference solution; an electrode; at least one chip on the surface whereof are integrated the ion-selective field effect transistors; a structure adhered on the first ion-selective field effect transistor configured to create a microreservoir on one gate of the first transistor, the microreservoir being filled with the reference solution; at least one microchannel which connects the microreservoir with the exterior, the at least one microchannel being filled with the reference solution; a substrate whereon is integrated the at least one chip, the connection tracks and the electrode; and, an encapsulating material which completely insulates the connection tracks and partially insulates the first and second ion-selective field effect transistor of the solution to measure.
 2. Ion sensor based on differential measurement, according to claim 1, wherein the reference solution is contained in a hydrogel.
 3. Ion sensor based on differential measurement, according to claim 1, wherein the first ion-selective field effect transistor is integrated in a first chip and at least one second ion-selective field effect transistor is integrated in a second chip.
 4. Ion sensor based on differential measurement, according to claim 1, wherein the first ion-selective field effect transistor and the second ion-selective field effect transistor are integrated in the same chip.
 5. Ion sensor based on differential measurement, according to claim 1, wherein the first and second ion-selective field effect transistor, the connection tracks, the electrode and a part of the measurement circuit are integrated in the same chip.
 6. Ion sensor based on differential measurement, according to claim 3, wherein the connection of connection points of the chip with the connection tracks is performed via wire bonding.
 7. Ion sensor based on differential measurement, according to claim 6, wherein the chips are encapsulated by a polymer, with the wires and the connection tracks being covered by the polymer and the gates of the first and second field effect transistor and the outlet of the microchannel being uncovered.
 8. Ion sensor based on differential measurement, according to claim 7, wherein the structure adhered on the first ion-selective field effect transistor is at least partially of a gas-permeable material and impermeable to the reference solution.
 9. Ion sensor based on differential measurement, according to claim 8, wherein the microchannel is a notch made in the chip whereon the first ion-selective field effect transistor is integrated.
 10. Ion sensor based on differential measurement, according to claim 1, wherein the microchannel forms part of the structure adhered on the first ion-selective field effect transistor.
 11. Ion sensor based on differential measurement, according to claim 10, wherein the microreservoir has a volume between 0.001 mm³ and 1 mm³, and the microchannel has a section of between 1 square micrometre and 10000 square micrometres and a length between 10 microns and 1 mm.
 12. Ion sensor based on differential measurement, according to claim 1, wherein the microchannel has removable and external sealing means to seal the content of the microreservoir and microchannel.
 13. Ion sensor based on differential measurement, according to claim 12, wherein the chips are manufactured from silicon on insulator.
 14. Conditioning receptacle for the ion sensor based on differential measurement defined in claim 13, wherein it is filled with the reference solution where the ion sensor is immersed based on differential measurement.
 15. Manufacturing method of the ion sensor based on differential measurement, described in claim 1, wherein it comprises the following phases: integrating a plurality of first ion-selective field effect transistors equally spaced on a first wafer; coupling a structure of bondable material on the first wafer, creating a plurality of equally spaced microreservoirs and microchannels in correspondence with the first ion-selective field effect transistors, so that each microreservoir is situated in correspondence and aligned with each first ion-selective field effect transistor; cutting the first wafer transversally creating chips, where each chip comprises a first ion-selective field effect transistor and a structure with a microreservoir and at least one microchannel; fixing on a substrate a chip, at least one second field effect transistor, the electrode and the connection tracks; connecting the connection tracks to the first and second field effect transistor and encapsulating the first and second field effect transistor and the connection tracks.
 16. Manufacturing method of the ion sensor based on differential measurement, according to claim 15, wherein it comprises adding a plurality of layers of bondable material subjected to a photolithography process on the first wafer, to generate the structure of bondable material with the microreservoirs and microchannels.
 17. Manufacturing method of the ion sensor based on differential measurement, according to claim 15, wherein it comprises previously subjecting the structure of bondable material to a recessing process for the creation of the microreservoirs and the microchannels.
 18. Manufacturing method of the ion sensor based on differential measurement, according to claim 15, wherein encapsulating material is additionally deposited on edges of the first and second ion-selective field effect transistor to electrically insulate the substrate of the first and second ion-selective field effect transistor.
 19. Manufacturing method of the ion sensor based on differential measurement, according claim 18, wherein different ion-selective membranes are fixed on the second ion-selective field effect transistors. 